Cell-Free Protein Synthesis (CFPS)
System Selection Guide
| System | Best For | Yield | PTMs | Disulfides | Cost |
|---|
| E. coli extract | Rapid prototyping, prokaryotic proteins | High (100-400 μg/mL) | None | Poor (reducing) | Low |
| E. coli PURE | Defined conditions, unnatural AAs | Medium (50-150 μg/mL) | None | Controllable | High |
| Wheat germ | Eukaryotic proteins, membrane proteins | High (100-500 μg/mL) | Limited | Moderate | Medium |
| Rabbit reticulocyte | Mammalian proteins, post-translational studies | Low (10-50 μg/mL) | Some | Poor | High |
| Insect (Sf21) | Glycoproteins, complex folds | Medium (50-100 μg/mL) | Glycosylation | Good | High |
| HeLa/CHO | Native mammalian proteins | Low (10-50 μg/mL) | Full mammalian | Good | Very High |
CFPS Troubleshooting Matrix
| Problem | Likely Causes | Design Fix | Reagent Fix |
|---|
| No expression | Rare codons at N-terminus, poor RBS | Codon optimize first 30 codons | Use BL21-CodonPlus extract |
| Low yield | Strong mRNA secondary structure, template issues | Optimize 5' UTR (ΔG > -5 kcal/mol) | Increase Mg²⁺ (10-18 mM), ATP |
| Aggregation | Hydrophobic protein, fast translation | Add solubility tags (MBP, SUMO) | Add 0.1% Tween-20, chaperones |
| Inactive protein | Misfolding, missing cofactors | Slow translation (use rare codons!) | Add GroEL/ES, DnaK/J |
| Truncation | Rare codon clusters, mRNA instability | Remove AGG/AGA/CUA clusters | Supplement rare tRNAs |
| Degradation | Proteolysis | N-terminal Met-Ala | Add protease inhibitors |
Codon Optimization for CFPS
Codons to Avoid in E. coli CFPS
| Codon | Amino Acid | Issue | tRNA Abundance |
|---|
| AGG | Arg | Very rare, stalling | 0.2% |
| AGA | Arg | Very rare, stalling | 0.4% |
| CUA | Leu | Low abundance | 0.4% |
| AUA | Ile | Rare | 0.5% |
| CGA | Arg | Inefficient decoding | 0.6% |
| CCC | Pro | Can cause pausing | 0.5% |
| GGA | Gly | Moderate | 1.1% |
Design Rules
- First 30 codons: Most critical - use only high-frequency codons
- Rare codon clusters: Avoid 2+ rare codons within 10 nt
- Rare codon content: Keep overall <5% of coding sequence
- GC content: Target 40-60% for balanced expression
- Avoid runs: No >6 consecutive G or C residues (secondary structure)
- Strategic slow codons: Place rare codons between domains (aids folding!)
When to Use Rare Codons
- Domain boundaries (allow cotranslational folding)
- Before complex structural elements
- When protein is prone to misfolding
mRNA Template Design
5' UTR Optimization
| Element | Optimal Design | Impact |
|---|
| RBS (SD sequence) | AGGAGG, 7-9 nt from start | Ribosome binding |
| Spacing | 7 nt between SD and AUG | Translation initiation |
| Secondary structure | ΔG > -5 kcal/mol | Accessibility |
| Upstream AUG | Avoid (causes false starts) | Reduces truncations |
Secondary Structure Targets
| Region | Ideal ΔG | Impact |
|---|
| -30 to +30 around AUG | > -5 kcal/mol | Translation initiation |
| Full 5' UTR | > -10 kcal/mol | Ribosome loading |
| RBS accessibility | Unpaired | Critical |
Template Format
| Format | Advantages | Disadvantages |
|---|
| Plasmid | Stable, high yield | Requires cloning |
| Linear PCR | Fast, no cloning | May need stabilization |
| mRNA | Direct translation | Unstable, expensive |
Disulfide Bond Formation
System Capabilities
| System | Native Disulfide Support | Additives Needed |
|---|
| Standard E. coli extract | Poor (DTT present) | IAM, PDI, GSSG/GSH |
| Oxidizing E. coli extract | Good | Pre-oxidized glutathione |
| Wheat germ | Moderate | Lower DTT, add PDI |
| PURE system | Minimal | Full oxidative system |
| Insect/Mammalian | Good | Microsome membranes |
Oxidative Folding Protocol (E. coli extract)
1. Deplete DTT from extract (dialysis or treatment with IAM 5 mM)
2. Add oxidized/reduced glutathione: 4 mM GSSG, 1 mM GSH (4:1 ratio)
3. Add 10 μM PDI (protein disulfide isomerase)
4. Optional: Add 5 μM DsbC (disulfide isomerase)
5. Express at 25°C (not 37°C) for better folding
6. Incubation time: 4-6 hours
Disulfide-Rich Protein Tips
- Start with wheat germ or oxidizing extract
- Use PURE system for precise control
- Consider co-expression of PDI/DsbC
- Verify by non-reducing SDS-PAGE
Expression Prediction from Sequence
| Feature | Good | Marginal | Bad |
|---|
| Rare codon content | <3% | 3-8% | >10% |
| First 30 codons rare | 0 | 1-2 | >2 |
| GC content | 45-55% | 35-45% or 55-65% | <30% or >70% |
| 5' UTR ΔG | > -3 kcal/mol | -3 to -8 | < -10 kcal/mol |
| Hydrophobic stretches | <5 consecutive | 5-7 | >8 consecutive |
| N-terminal residue | Met-Ala, Met-Ser, Met-Gly | Met-Val, Met-Thr | Met-Arg, Met-Lys |
| Cysteine pairs | Paired (even number) | Mixed | Odd number (free thiols) |
Solubility Enhancement Strategies
Fusion Tags (ranked by effectiveness)
| Tag | Size | Solubility Enhancement | Cleavage | Notes |
|---|
| MBP | 40 kDa | Excellent | TEV, Factor Xa | Best overall |
| SUMO | 11 kDa | Very Good | SUMO protease | Native N-terminus after cleavage |
| NusA | 55 kDa | Excellent | - | Large size |
| Trx | 12 kDa | Good | Enterokinase | For disulfide proteins |
| GST | 26 kDa | Moderate | - | Dimeric |
| His₆ | 1 kDa | Minimal | - | Mainly for purification |
Buffer Additives for Solubility
| Additive | Concentration | Mechanism |
|---|
| Trehalose | 50-100 mM | Chemical chaperone |
| Glycerol | 5-10% | Reduces hydrophobic aggregation |
| L-Arginine | 50-100 mM | Suppresses aggregation |
| Tween-20 | 0.05-0.1% | Prevents surface adsorption |
| Proline | 50 mM | Osmolyte stabilization |
Chaperone Supplementation
| Chaperone System | Target Problem | Concentration |
|---|
| GroEL/GroES | General folding | 1-2 μM |
| DnaK/DnaJ/GrpE | Aggregation-prone | 1 μM each |
| Trigger Factor | Nascent chain | 1-2 μM |
| ClpB | Aggregate resolubilization | 0.5 μM |
Temperature Optimization
| Temperature | Use Case | Trade-offs |
|---|
| 37°C | Fast expression, stable proteins | Higher aggregation risk |
| 30°C | Balanced (default) | Good compromise |
| 25°C | Disulfide proteins, complex folds | Slower, better folding |
| 18-20°C | Aggregation-prone proteins | Much slower, best folding |
| 16°C | Cold-shock proteins | Very slow, specialized |
E. coli Extract Preparation (Key Variables)
| Variable | Impact | Optimal Range |
|---|
| Cell density at harvest | Ribosome content | OD₆₀₀ 2.5-3.5 |
| Lysis method | Extract activity | Sonication, bead beating |
| Run-off reaction | Removes endogenous mRNA | 20-80 min at 37°C |
| Mg²⁺ concentration | Translation fidelity | 10-18 mM |
| K⁺ concentration | Translation rate | 150-200 mM |
| Energy system | Sustained synthesis | ATP/GTP, creatine phosphate |
PURE System Specifics
Advantages
- Defined composition (no proteases/nucleases)
- Linear DNA templates work well
- Unnatural amino acid incorporation
- Reproducible between batches
Limitations
- No chaperones (add separately)
- No post-translational modifications
- Lower yields than crude extracts
- Higher cost
When to Use PURE
- Unnatural amino acid incorporation
- Studying translation mechanisms
- "Clean" proteins needed
- Protease-sensitive targets
- Linear template expression
Common Artifacts and Solutions
Low Molecular Weight Bands
Causes: Premature termination, proteolysis, internal initiation Solutions:
- Optimize rare codon clusters
- Add protease inhibitors
- Check for internal AUG codons
- Use PURE system
Higher MW Bands
Causes: Incomplete termination, read-through, aggregation Solutions:
- Ensure strong stop codon (UAA preferred)
- Check template 3' end
- Add release factors (RF1/RF2)
- Reduce protein concentration
No Soluble Protein
Causes: Aggregation during synthesis Solutions:
- Lower temperature (25°C → 18°C)
- Add chaperones
- Use solubility tag
- Optimize translation rate
References
CFPS Overview
Extract Preparation
PURE System
Wheat Germ
Codon Optimization
Disulfide Formation
Solubility Tags
Temperature Effects
